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Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation
Jennifer S Carew1, Ernest C Medina, Juan A Esquivel
1Institute for Drug Development, University of Texas Health Science Center, San Antonio, TX 78245, USA.
Abstract:
Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin-conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ-induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ-induced aggresome formation, but promoted CQ-mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR-mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR-induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ-induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ-mediated aggregate formation, superoxide generation and apoptosis.
Insights
Inhibiting autophagy with chloroquine (CQ) causes protein buildup, enhancing cancer cell death when combined with vorinostat (VOR). This CQ/VOR combination also reduced tumor growth in models, highlighting a new therapeutic strategy for colon cancer.
Area of Science:
- Cellular Biology
- Cancer Research
- Molecular Medicine
Background:
- Autophagy is a cell survival pathway crucial for nutrient recycling, particularly in the tumor microenvironment.
- Autophagy can promote cancer cell survival and resistance to chemotherapy by providing alternative energy sources.
- Disrupting autophagy is a potential strategy to overcome treatment resistance in cancer.
Purpose of the Study:
- To investigate the effects of combining autophagy inhibition (chloroquine) with HDAC inhibition (vorinostat) on colon cancer cells.
- To determine the mechanisms underlying the combined effects of chloroquine and vorinostat on protein accumulation and apoptosis.
- To evaluate the therapeutic potential of the chloroquine/vorinostat combination in a colon cancer xenograft model.
Main Methods:
- Autophagy inhibition using chloroquine (CQ) and assessment of ubiquitinated protein accumulation.
- Treatment of colon cancer cells with CQ and vorinostat (VOR), alone and in combination.
- Analysis of apoptosis, aggresome formation, and superoxide generation.
- Evaluation of tumor burden and apoptosis in a colon cancer xenograft model.
Main Results:
- CQ-induced autophagy disruption led to lysosomal accumulation of ubiquitinated proteins, distinct from proteasome inhibitor effects.
- The combination of CQ and VOR synergistically enhanced apoptosis in colon cancer cells.
- CQ/VOR treatment significantly increased superoxide generation, which was essential for cell death and aggregate formation.
- Combined CQ/VOR therapy reduced tumor burden and increased apoptosis in vivo.
Conclusions:
- Inhibition of autophagy with CQ potentiates VOR-mediated apoptosis in colon cancer.
- The CQ/VOR combination induces ubiquitinated protein accumulation, superoxide generation, and cell death.
- Targeting autophagy in combination with HDAC inhibition represents a promising therapeutic strategy for colon cancer.
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